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After washing four times with PBST, add the substrate and read the absorbance at 405 nm. Indirect immune fluorescence-Using 2 slides according to the manufacturer\'s instructions, ANA was determined by indirect immune fluorescence (IIF)assay. Dilute the serum with PBS (1: 40, 1: 80, 1: 160 or 1: 320). The diluted samples were added to the slide, incubated for about 40 min and washed with PBS.

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